Fluid coverage sensing system and method

ABSTRACT

A system for applying a fluid to a substrate bearing a sample for analysis has an array of sensor plates positioned to sense the presence of fluid in contact with respective areas of the substrate. In a particular embodiment, fluid presence in different areas of the substrate is sensed by the effect of the fluid and its identity on the impedances of capacitors formed between sensor plates within the array. In a more particular embodiment, by polling the sensor array continually while fluid is applied to the substrate determine a coverage map, a fluid dispensing mechanism can be controlled to efficiently cover the entire substrate with fluid a minimal amount of fluid, thereby reducing waste.

RELATED APPLICATION DATA

This is a continuation of International Patent Application No.PCT/EP2016/079547 filed Dec. 2, 2016, which claims priority to and thebenefit of U.S. Provisional Application No. 62/262,855, filed Dec. 3,2015. Each of these prior patent applications is incorporated byreference herein.

FIELD

This disclosure relates to the general field of systems in which aliquid is applied to treat a sample for study or analysis, and moreparticularly to systems and methods in which a sample is placed on aslide and then covered with a liquid that aids in its analysis.

BACKGROUND

In a number of systems used in medical diagnostics and other biologicaltechnologies that are used to test or study a biological sample (forexample, a thin sample of tissue or smear of cells), the sample isplaced on a glass slide or other substrate, and a fluid is applied overit. The fluid typically contains one or more chemicals that interactwith the sample, for example, staining the tissue sample or binding tospecific analytes in the sample. The treated sample is then reviewed byhuman observation or possibly using automated sensors to determinewhether the sample exhibits one or more properties after interactionwith the fluid.

For proper analysis of the sample, it is normally necessary for thefluid to be applied to cover at least a sufficient portion of the sampleor the entire sample or possibly the entire surface of the slide.Covering the sample or the slide may be difficult to do reliably due tothe variability of the surface forces at the fluid-glass boundary, andpossibly due to errors in automated dispensing of fluids to the sample.As a result, the sample or slide may only be partly covered by thefluid, or a bubble may be formed separating the sample from the fluid,either of which may result in an inaccurate analysis of the sample.

Most commonly, the application of the fluid is performed by an apparatusthat automatically applies the fluid to the slide inside an instrument.This makes inspection or monitoring by a human technician of the puddlecoverage and the lack of bubbles difficult, and such constant monitoringby a person defeats the purpose of automation.

In the absence of human visual inspection, a number of approaches toensure the full sample or slide coverage have been employed. Thoseapproaches include flooding the slide with the fluid, dispensing a largepuddle of the analysis fluid, or repeatedly supplying fluid to theslide. However, all such approaches are wasteful of the fluid used inthe analysis, with the result that processing of the tissue is moreexpensive.

SUMMARY

A system is disclosed for detecting fluid coverage of a substrate, thatincludes a substrate holder for holding the substrate, an array ofcapacitive plates in proximity to the substrate holder, a fluid supplyconfigured to deliver a fluid to the substrate held on the substrateholder, and sensing electronics in electrical connection to the array ofcapacitive plates, wherein the sensing electronics periodically connecttwo poles of a current to at least one pair of plates and detects anoutput signal indicative of an electric property between the at leastone pair of plates. In particular embodiments, the disclosed systemautomatically detects and confirms sufficient coverage of a sample orthe substrate in an analysis or treatment with the fluid. In otherparticular embodiments, the disclosed system provides a quantitativeassessment of the coverage of the fluid puddle on the slide inside theinstrument used, either by an automated coverage verification system orusing an integrated sensor array connected with an embedded feedbackloop that controls the dispensing and/or mixing of the fluid onto thesubstrate during fluid application to the substrate, such as forstaining of a biological sample held on the substrate. In still otherparticular embodiments, the disclosed system provides active,non-invasive, real-time detection of the volume of fluid that has beendispensed onto the slide, which, for example, could be used as atroubleshooting system in development or technical support service ofthe system. In even other particular embodiments, the disclosed systemcan provide identification and/or verification of the type of fluid(such as a dye solution like eosin, a buffer solution, a non-polarsolvent) that is dispensed to the substrate.

Also disclosed is a method including providing a sample support having asample thereon to be treated with a reagent fluid, applying the reagentfluid to the sample support, and, sensing the presence of the reagentfluid on the sample support, wherein sensing includes detecting arespective impedance for at least one pair of a plurality of sensorelements positioned in proximity to the sample support such thatpresence of the reagent fluid in a respective subarea of the samplesupport alters the impedance of a capacitor formed between the at leastone pair of the plurality of the sensor elements. In particularembodiments, the sample is a biological sample, for example a tissuesection. In other particular embodiments, a degree of fluid coverage isdetermined from said sensing. In further embodiments, applying thereagent fluid includes applying the reagent fluid from a fluid supplymechanism that is in feedback control with the sensing step such thatthe reagent fluid is dispensed, mixed or otherwise dispersed to achievea predetermined degree of fluid coverage on the sample support.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of disclosed system and method willbecome apparent in view of the Detailed Description that follows, andthrough reference to the accompanying figures in which:

FIG. 1 is perspective diagram of an embodiment of a disclosed slidetreatment system.

FIG. 2 is a plan view of a slide with an exemplary sample and a partialpuddle of fluid spread thereon.

FIG. 3 is an exploded perspective view of a slide treatment systemaccording to the disclosure including a capacitive sensor array fordetecting the nature and distribution of a liquid on a substrate.

FIG. 4 is a detailed vertical cross-sectional view through a transparentslide and to particular embodiment of an array of capacitive platesbelow.

FIG. 5 is a plan view of a substrate holder of a disclosed substratetreatment system showing and embodiment of a capacitive sensor arraybelow and how electric fields extend from the capacitive array into andthrough the substrate to a biological sample and liquid above.

FIG. 6 is a schematic diagram of an embodiment of an exemplarycapacitive sensor array according to the disclosure.

FIG. 7 is a schematic diagram of the components of a system according toa disclosed embodiment.

FIG. 8 is a flow chart illustrating an exemplary logic operation for acomputerized control system for substrate treatment according to thedisclosure.

FIG. 9 is a perspective view schematic diagram of an alternativeembodiment in which the capacitive sensor array is in the substrateitself, rather than the holder.

FIG. 10 is a graph showing voltage changes as a function of AC frequencyfor various types of fluids.

DETAILED DESCRIPTION

Disclosed is a system for detecting fluid coverage of a substrate thatincludes a substrate holder for holding the substrate, an array ofcapacitive plates in proximity to the substrate holder, a fluid supplyconfigured to deliver a fluid to the substrate held on the substrateholder, and sensing electronics in electrical connection with the arrayof capacitive plates. According to one embodiment, the sensingelectronics periodically connect two poles of a current to at least onepair of plates and detects an output signal indicative of an electricproperty between the at least one pair of plates. The array ofcapacitive plates can be one or more of integrated into the substrateholder, integrated into the substrate and held in a position on a sideof the substrate opposite the substrate holder. For example, inparticular embodiments, the substrate is a microscope slide and thearray of capacitive plates is contained in a structure supporting theslide, is contained in the slide itself, or is supported above the slideso as to directly sense the fluid on the slide, such as a puddle offluid on the slide.

In particular embodiments, the sensing electronics are configured todetermine a baseline output signal indicative of a substrate having nofluid coverage thereon. In other particular embodiments, the sensingelectronics are configured to develop a coverage map of the fluid on thesubstrate, such as it is dispensed, mixed, moved or removed on or fromthe substrate by detecting a difference in the electric field due to thepresence of the fluid in the vicinity of a plurality of pairs ofcapacitive plates in comparison to the baseline output signal indicativeof the substrate having no fluid coverage. In more particularembodiments, the coverage map is provided to a controller that controlsthe fluid supply, thereby establishing a feedback loop that directs thefluid supply to apply additional fluid to the substrate until apre-determined coverage of the substrate is reached. In a similarfashion, the coverage map can be used to causes the system to mix,distribute or remove the fluid as needed for a particular process step.

While any source of current can be used in the sensing electronics ofthe disclosed system, in particular embodiments the current is an ACcurrent, and the AC current can be scanned over multiple frequencies toaid in detection of the identity of the fluid applied to the substrate.Various electric and physical properties can be detected betweencapacitive plates in order to detect and/or identify a fluid applied toa substrate, and these electric and physical properties include forexample, one or more of voltage, current, capacitance, electric fieldstrength, dielectric constant and impedance.

In another particular embodiment, the sensor area including the array ofcapacitive plates, includes a plurality of sensors each directed to arespective area of the slide, with each sensor comprising a capacitorstructure having spaced conductors between which an electric field isformed when current is applied across the spaced conductors or one ofthe conductors is charged by current, wherein an electrical fieldextends through the slide such that the fluid, when present on the slidein the particular area, affects electrical properties of the capacitorstructure.

According to another particular embodiment, a disclosed system includesa slide or other sample support having thereon a sample of material fortreatment or analysis, the treatment or analysis including applicationof a fluid to the sample. The system according to this embodimentfurther has a cradle for receiving the slide, and a fluid supplysupported so as to supply the fluid onto the slide. The fluid supply iscontrolled by control circuitry. A sensor set is included, which sensorset includes a plurality of sensors, each sensor set directed to arespective area of the slide, and each sensor set representing acapacitor structure having spaced conductors between which an electricfield is formed when current is supplied to the spaced conductors. Theelectrical field generated by each sensor set can extend into the slide,into the sample, and even into the fluid, when present on the slide inthe area, affects electrical properties of the capacitor structure. In aparticular embodiment, the conductors are fabricated as planar squareplates, wired into linear row and column arrays, creating a spatiallyaddressable sensing area. In another embodiment, conductors are wiredtogether diagonally, to create a different shape to thespatially-selectable sensing area. In still another embodiment, a singlegrid is the reference signal for individually addressable sensing sets.In still another embodiment, the plates may be located at differentdepths within the supporting structure, either with two-dimensionaladdressing of linear arrays to form a spatially selective sensingsignal, or with individual sensing plates referenced to a singlebackplane.

As in the embodiments above, the sensor set(s) can be in a structuresupporting the slide or sample support, contained in the slide or samplesupport, or may be supported elsewhere in the apparatus, such as abovethe sample or between the sample and the slide, provided that it ispositioned so that it can sense the presence of fluid on the slide. Whena sample on a slide is placed in the apparatus, interrogating circuitryin a single polling, or a continuous or periodic polling, supplies anelectrical current to the electrodes of each sensor and detects one ormore electrical properties of the capacitor formed via the establishmentof an electric field through the slide, the sample, and/or the fluidsample (or air if no fluid is present).

According another particular embodiment, the electrical circuitry isconfigured to derive coverage assessment data for the slide from arespective output signals responsive to the application of aninterrogating signal or current to each sensor that is reflective of arespective degree of fluid coverage in the vicinity of the sensor andthereby generate a coverage map. In a further particular embodiment, acontrol loop controls the fluid supply (or a mechanism that spreads thefluid on the slide or some other mechanism redistributing fluid on theslide, such a vibrating support) is responsive to the coverage map so asto cover the entire slide, or a predetermined desired coverage areathereof, with fluid or to ensure adequate coverage of the sample.

In still further particular embodiments, the electrical circuitry makesa determination of the identity of a fluid based on the electricalchanges, wherein the current applied to the sensors includes a pluralityof currents each of a distinct respective frequency, and thedetermination of the identity of the fluid relies on a trained logisticregression algorithm supported by the electrical circuits. Theelectrical circuitry can also determine a volumetric assessmentcorresponding to an amount of fluid on the slide based on the coverageassessment and the identification of the fluid. In another embodiment, asystem is disclosed that includes a sample support structure supportinga sample thereon for treatment with a fluid.

In another particular embodiment, a system is disclosed wherein thesample support structure is a slide having thereon a sample of materialfor treatment or analysis, which treatment or analysis involvesapplication of fluid to the sample. In this embodiment, the systemfurther includes a cradle receiving the slide, a fluid supply supportedso as to supply the fluid onto the slide; and a mixing or dispersingapparatus that agitates or otherwise spreads or moves the fluid once onthe slide (such as an opposable surface, an air knife, a moveablespreading device that forms a capillary gap between the slide and thespreading device, a source of vibrational energy or acoustic energy tocause mixing, or the like). The fluid supply and the mixing ordispersing apparatus are controlled by the control circuitry, forexample, in a feedback loop in communication with a fluid coverage map,to ensure fluid is located, placed, removed or otherwise manipulated toensure that treatment of the sample is conducted according to apre-determined protocol.

Also disclosed is a method, the method including providing a samplesupport having a sample (for example, a biological sample, such as atissue section) thereon to be treated with a reagent fluid, applying thereagent fluid to the sample support and sensing the presence of thereagent fluid on the sample support. The sensing step can includedetecting a respective impedance for at least one pair of a plurality ofsensor elements positioned such that presence of the reagent fluid in arespective subarea of the sample support alters the impedance of acapacitor formed between the at least one pair of the plurality of thesensor elements. In more particular embodiments, a degree of fluidcoverage is determined from said sensing, which in even more particularembodiments is used to control application of the reagent fluid byapplying the reagent fluid from a fluid supply mechanism that is infeedback control with the sensing step such that the reagent fluid isdispensed, mixed or otherwise dispersed to achieve a predetermineddegree of fluid coverage on the sample support.

In another embodiment, the disclosed method includes providing a samplesupport (such as a microscope slide) having a sample thereon that is tobe treated with a reagent fluid, applying the reagent fluid to thesample support, and sensing presence of the reagent fluid on the slide.According to this embodiment, sensing includes detecting the respectiveimpedance (understood to include those properties that compriseimpedance including capacitance, resistance, dielectric strength, andconductivity) for each of a plurality of sensor elements positioned suchthat presence of the reagent fluid in a respective subarea of the slidealters the impedance of a capacitor formed within the sensor element. Ina particular embodiment, the method further includes determining adegree of fluid coverage from the sensing, for example, to provide acoverage map. The method can further include application of the reagentfluid using a fluid supply mechanism operated by electrical circuitry,mixing or dispersing of fluid once on the slide using a mechanismoperated by electrical circuitry, and/or controlling the fluid supply,mixing, or dispersion mechanisms using data derived from the detectingof the impedance of the sensor elements so as to cover a predeterminedcoverage area of the slide with the reagent fluid. In even moreparticular embodiments, an identification of the reagent fluid is madebased on the detecting, detection of bubble formation, and/or detectionof reagent fluid evaporation can further be made by the detecting of theimpedances.

According to still another embodiment, the disclosed method includesproviding a sample support (such as microscope slide) having a samplethereon to be treated with a reagent fluid, applying the reagent fluidto the sample support, and sensing presence of the reagent fluid on theslide. The sensing in this embodiment includes detecting respectiveimpedances for each of a plurality of sensor elements positioned suchthat presence of the reagent fluid in a respective subarea of the slidealters the impedance of a capacitor formed by the sensor elements.Detecting the impedance may be done by applying an electrical current toone of the plates of the capacitor formed by the plates of a sensorelement and receiving a signal at the other of the plates, which may bein the form of an output current from the other plate and from whichimpedance may be determined by the drop in voltage or the drop inamperage. Also a degree of coverage may be determined from the sensing.The step of applying the reagent can then be performed using a fluidsupply mechanism operated by electrical circuitry, and the method canfurther include controlling the fluid supply mechanism (or other meansof affecting coverage) using data derived from the detecting of theimpedance of the sensor elements so as to cover a predetermined coveragearea of the slide with the reagent fluid. In more particular embodiment,the method further includes detecting bubble formation in the reagentfluid by detecting the impedances.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise.

The terms “comprising,” “including,” “having,” and the like are usedinterchangeably and have the same meaning. Similarly, “comprises,”“includes,” “has,” and the like are used interchangeably and have thesame meaning. Specifically, each of the terms is defined consistent withthe common United States patent law definition of “comprising” and istherefore interpreted to be an open term meaning “at least thefollowing,” and is also interpreted not to exclude additional features,limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary unless a particular order is clearlyindicated by the context.

As used herein, the term “about” refers to plus or minus 1-10% of thereferenced number, for example plus or minus 1-5% of the referencednumber, such as plus or minus 1-2% of the referenced number.

As used herein, the term “substantially” refers to at least 90%, forexample at least 95%, such as at least 99% of the referenced object ofthe term.

As used herein the term “biological sample” refers to a sample obtainedfrom an organism or otherwise derived from an organism's tissues or fromfluids present in or expelled from the organism.

Referring now to FIG. 1, in the system of one embodiment, a glassmicroscope slide 1 has applied to it a thin sample 3 of tissue or othermaterial to be analyzed. The slide itself is commonly made of glass, butmay also be of a variety of materials and configurations, and withvarious coatings. The length and breadth of the slide may vary widely,but the most commonly used slide 1 has dimensions of about 1 inch byabout 3 inches (or about 25 mm by about 75 mm). The thickness of theglass slides is usually in the range of about 0.9 to about 1.2 mmNormally, the tissue sample is extremely thin, such as with a thicknessin the range of about one micron to about 20 microns. Other types ofsample support structures other than slides, such as plates ortest-strips or Petri dishes, may be employed according the disclosure.

Apparatus and Holder Structure

The slide 1 is placed or conveyed to an apparatus generally indicated at5 where it is supported on a holder structure 7. Holder structure 7 hasan upper surface on which the slide 1 rests, and may optionally have acradle or tray structure that holds the slide 1 in place on it. Inaddition, the holder structure 7 may include temperature controls, suchas a heater, to bring the temperature of the slide 1 and the sample 3 toan appropriate temperature for the process to which it is subjected.

A fluid supply mechanism 9 provides fluid to the top of the slide 1 andover the sample 3. This fluid supply mechanism 9 is controlled byelectronics, such as a computer or processor of the apparatus 5, so asto control supply of the fluid to cover the slide 1.

Referring to FIG. 2, in the preferred embodiment shown, as the fluid isapplied to the slide 1, it forms a puddle 11. The size of the puddledesired may vary depending on the application. Ordinarily, the puddleshould cover the at least the sample, although there may be applicationsin which only a predetermined coverage area constituting only part ofthe sample is covered with the given fluid. In other scenarios, it isdesirable to cover the entire slide so as to be certain that the entiresample is covered. In those applications, when the puddle 11 does notcover the entire slide, more of the fluid is supplied (orredistributed), and the puddle 11 expands as the fluid is supplied untilit covers the entire upper surface of the slide, as will be describedherein.

In other embodiments, a different type of fluid supply mechanism 9 maybe used to apply two or more different fluids across the sample and theslide in generally parallel stripes, swaths or lanes, wherein each fluidhas a respective desired coverage area. The process herein may also beused with such a fluid-supply system advantageously to ensure properapplication of the various fluids to the slide.

Referring to FIG. 3, the holder 7 has a base structure 13 with an upperreceiving area 15 onto which a sensor array plate 17 is mounted, fromwhich mounted position the plate 17 is electrically connected to theelectronics of the apparatus (not shown).

Sensor Array

Referring to FIG. 4, sensor array plate 17 has a matrix of generallyplanar capacitance pads or plates 19 with upwardly facing surfaces. Inthe preferred embodiment, the plates 19 are each approximately 3 mm×3 mmsquare, but differently sized plates may be used, for example, that arefrom about 2 mm to about 4 mm square, and in different shapes, such asrectangular shapes. Furthermore, the spacing between the plates can bevaried to provide differing levels of detection within the substrate aninto a fluid placed thereon, for example, from about 0.05 mm to about0.2 mm, for example, about 0.1 mm. In addition, the sensor matrix of thepreferred embodiment shown is 8 rows of 16 plates each, but the numberof rows and columns may be varied, and the configuration of thearrangement may be something other than a rectangular matrix as well.

The plates are adjacent to, but electrically separate from, each other,except for the wiring between the plates shown in FIG. 6. The sensorplate 17 fits into or is mounted on the base 3, and electricalconnections or contacts connect the plates 19 of the sensor plate 17 toelectrical circuitry in the base 3 that allows the electronics of theapparatus to access the sensors plate 17 for determining the presence ofliquid or other material on the slide. The sensor plate 17 may fit intoan upper recess, such as the recess shown in FIGS. 3 and 4 betweenvertical walls 18, which define a sort of cradle that holds the sensorplate 17 in place. Walls 18 may extend above the sensor plate 17 to holdthe slide 1 thereon in place so as to predictably align and position theslide 1 with respect to the sensor plate 17. It will be understood thata variety of structural arrangements can be employed to accomplish thealignment of the slide and the sensor plate 17.

When the slide 1 rests on the sensor plate 17, the sensor plate 17 canbe employed to detect the fluid applied to the slide by the fluid supplymechanism 9 of FIG. 1.

Optionally, a protective layer or thin plate of material may be usedoverlying the plates 19, provided that the protective layer does notprevent the sensing of fluid on the slide.

Sensor Operation

The sensor plate 17 operates similarly to a capacitive touch screen on acell phone, as is illustrated in the detail diagram of FIG. 5.

Sensing electronics 21 are connected with the sensor plate 17, andselectively supplies electrical power to the plates 19 to scan thecapacitive properties of each plate 19. To scan a given plate 19 a, thesensing electronics 21 periodically connect two poles of a current,preferably an AC current at a voltage of from 1 to 10 volts, to plate 19a and one or more adjacent plates 19 b. When so connected, plate 19 aand the adjacent plates 19 b act as a capacitor, forming an electricalfield 23 between the plates 19 a and 19 b.

As the fluid is applied to the slide 1, the puddle 11 eventually extendsinto the effective area of the electrical field of capacitor plate 19 ain the sensor plate 17. The electrical field is illustrated by phantomlines 25, and it extends from one plate 19 a, through the slide 1, andthrough the sample 3 and the puddle 11 when they are on the slide 1 inan area near the plate 19 a, and then to the other plates 19 b,completing the capacitor circuit.

There may be a layer of dielectric material over the matrix of plates,but also both the puddle 11 and the sample 3 have dielectric propertiesto some degree. The puddle fluid may be aqueous, although for sometissue analysis it may be organic or other types of fluid, and thematerial of the sample 3, while very thin, may be any sort of organictissue or material. As a result, the presence of the puddle 11, and tosome lesser degree the sample 3, alters the impedance between the plates19 a and 19 b, and alters properties of the output signal producedresponsive to the electric field established through the puddle byapplication of the input current or signal.

The sensing electronics 21 detect electrical properties of the electricfield established across the capacitor 19 a/19 b and transmit dataindicative of those properties or some comparative data derived fromthose detected values to the other circuitry of the apparatus, as willbe described below. A comparison of the properties of the output signalwhen the plates 19 a and 19 b are connected to the current source ascompared to a baseline impedance of the slide 1 alone with only air overit, and no sample or fluid present allows a determination of theimpedance created by the puddle and/or the sample, from which a numberof conclusions may be drawn, as will be described below.

It will be understood that a system according to the invention mayemploy one or more of a variety of designs to determine the impedance orcapacitance or a related property of the sensors other than simplypassing a current through the respective plates.

Sensor Plate

FIG. 6 shows a schematic of the electrical connections between theplates 19 in the sensor plate 17 and leads that are connected with thesupporting electrical circuitry 21.

According to the one embodiment, all of the plates 19 are coplanar andorganized in eight columns and sixteen rows. Each of the sixteen rowshas a respective driving line 22 associated with it, and each column hasa respective sensing line 24 associated with it. The driving lines 22and the sensing lines 24 are connected with the sensing electronics 21(FIGS. 5 and 7), which selectively applies input current to establish anelectric field with respect to each of the plates 19 in a spatialencoding scheme.

As seen in FIG. 6, the internal connections of the plates 19 in thearray are that the plates 191 (the white squares) in each row areconnected in series to a respective driving line 22, and alternate withplates 192 (the shaded squares) of the columns. Plates 192 of eachcolumn are connected in series to a respective sensing line 24. Withthese connections, it is possible to poll each plate 19 by feeding aninput current and detecting the output signal between the lines 22 and24 of the row and column of that particular plate.

System Operation

Referring to FIG. 7, the circuitry and functionality of the apparatususing the sensor plate 17 are illustrated.

The sensor array 17 comprises a dielectric layer 25 and the specificsensors 27 of the array which generally refer to each respective plate19. The sensor array 17, together with the target sample fluid interface29 which generally comprises the slide 1, the sample 3 and the puddle11, together form a set of capacitors, as described above.

The apparatus according to one embodiment is a computerized system thatincludes one or more microprocessors and electronic data storageconnected with the microprocessors. The electronic data storage storesdata that constitutes software instructions executed by themicroprocessor or microprocessors so as to control operation of theapparatus, as is well known and common in the art.

The individual plates 19 of the sensor array 17 are accessed by sensinghardware 31, which corresponds to the electronics 21 in FIG. 5. Thesensing hardware 31 includes an excitation circuit 33 that producescurrent, preferably AC current with a voltage in the range of 1 to 10 ormore volts. The current produced in some embodiments includes one ormore AC currents, each with a respective pre-determinedalternating-current frequency, for identification of the fluid on theslide, as will be discussed below.

The excitation circuit 33 supplies the current to signal multiplexingcomponent 35, which is essentially a switching component controlled by acontrol and synchronization component 37. The control component 37 iscontrolled by the computerized apparatus, preferably by a microprocessorin the apparatus operating according to stored control software datastored in computer accessible memory. Signal multiplexing component 35controls switches so that it communicates the current from theexcitation circuit 33 in series to each of the various sensors 27 of thesensor array 17, which are all polled each polling cycle. The pollingtakes place periodically and continually, with a duty cycle appropriateto the operation of the apparatus, e.g., every 1 to 20 seconds.

In the system and method shown, the 128 individual plates or sensors ofthe sensor array 17 are each individually polled in each polling cycleby control component 37 causing the signal multiplexing component 35 toconnect the AC current to the relevant lines 22 or 24 for each givenplate 19, and to measure the output signal from the relevant line 22 or24 on the other side of the capacitor structure created when each plate19 is polled.

The signal multiplexing component 35 receives the output signal from theindividual sensors 27 being polled, and supplies this signal to a sensordetect circuit 39. The sensor detect circuit 39 receives the outputcurrent from signal multiplexing component 35 and also a reference inputsignal from excitation circuit 33 so as to compare these two and makedeterminations of the relative electrical properties, which may include,for example, amplitude or amperage, voltage, and phase shift of the A/Csignal that are imparted by the given capacitor.

The main apparatus processor system is indicated at 40. The data fromthe detect circuit 39 of the sensing hardware 31 is transmitted to asignal processing component 41 operated by the processor executingsoftware stored so as to be accessible electronically thereto. Theprocessor stores at least some data from earlier readings, and uses thestored data as well as the present polling data regarding the sensorarray to determine the coverage of the slide by the fluid, and possiblyto determine the type of fluid applied to the slide and to determine thevolume of fluid on the slide.

The signal processing process step involves processing the data of thepolling of the sensors to derive information about the slide and thefluid or the sample thereon, which may be one or more of a variety ofsorts of information, such as, e.g., coverage of the slide by fluid, aswill be discussed below. Whatever the information derived may be, theresult of the signal processing is provided as answer or output 43.

The processor or internal computer of the apparatus 5 executes storedsoftware that causes it to function according to the method shown in theflowchart of FIG. 8.

Initially, to start the process, the system is turned on and the sensorplate or array is positioned in the apparatus 5 or stainer in step 49and the slide is placed on it. An initial poll of the sensor array withthe slide and sample, but no fluid applied yet, is performed in step 51.Alternatively, an initial poll can be performed at a frequency thatcreates an electric field that measures only electrical properties ofthe slide. The capacitor plates 19 each have a respective output currentthat is analyzed for an electrical property that is indicative of thechange in impedance due to the application of fluid to the slide in thearea of that particular plate 19. This yields data that serves as abaseline value for each plate of the sensor array. In the preferredembodiment, the electrical property used to determine coverage of theslide in the area of a given plate is the voltage drop with respect toan adjacent plate 17 when a drive current is supplied to the plates 17.

At decision 53, the sensor plate is polled to derive a current sensorvalue for each plate 19 derived from one or more detected electricalproperties of the input and output signals across each sensor plate 19,such as, e.g., the output voltage or the ratio of output to inputvoltage. This current sensor data for each plate is compared at decision53 with the baseline threshold level of the given plate as determined instep 51. If the current sensor data indicates that the level ofimpedance for the plate 17 has remained constant, i.e., there has beennot enough fluid added to the slide to cause a change in impedance, thendecision 53 loops to the next duty cycle, and it keeps on looping andpolling the sensor array until the currently polled sensor array dataindicates that the fluid is present on some of the plates 18. Thisindicates that the puddle 11 is beginning to be formed on the slide 1.

Coverage Map

Once the puddle 11 begins to be formed, the system scans the sensor orarray 17 and identifies those plates 19 of the sensor array 17 that,based on a comparison of their current sensor data to the baseline data,have fluid partly or completely overlying them.

The specific differences between the output and input signals applied tothe individual capacitors 19 of the sensor array 17 are usually aconstant value until the fluid is applied to the slide. When the fluidis initially applied to the slide, if there is only partial coverage ofa given area of a plate, there is only a slight increase in the outputsignal compared to the input signal due to a fractional change of theeffective dielectric strength of the medium between the plates. For thedetermination of coverage to be complete, this output signal valueshould exceed some pre-determined threshold level to ensure that thearea of the plate 19 is entirely covered by the fluid, and also toensure that there are no bubbles or other interruptions in the fullapplication of the puddle 11 to the slide and sample, such as where anoil drop is trapped below an aqueous puddle, which would result in anunusually low output signal due to the non-polar nature of the oil inthe drop.

For the preparation of the coverage map, the processor and associatedelectrical circuitry continually and repeatedly check the impedance orother relevant electrical property of each of the pads 19 on the sensormatrix 17. The resulting electrical data for each plate 19 is recordedand stored in a respective element of an array of electronicallyaccessible stored data values stored in the system. When there is achange in the impedance for any particular pad or plate 19 that exceedsa pre-determined threshold difference or ratio with respect to theprevious impedance value, the value of the corresponding element in thearray of data values or flags has the value for that particular plate 19set to a value that indicates that it is covered with the puddle. Thisarray of coverage map values is modified in real time as the values ofthe impedances of each of the plates 19 changes. Each element of thecoverage map array and corresponding display may be a simple data flagor indication that the respective area is covered with fluid.Alternatively, the elements of the array may be data values indicativeof the degree of coverage over a range from no fluid present to totallycoverage with fluid.

The values of this array are used by the control system to determinewhether to direct the fluid supply mechanism 9 to supply more fluid tothe slide, to discontinue fluid supply, or to mix or disperse the fluidfurther on the slide with a distributing device in the system until allof the elements of the array indicate that the slide is completelycovered, or when a set of the elements of the array corresponding to adesired predetermined coverage area, e.g., the shape of the sample,indicate that the area is covered with fluid, and that there are nobubbles. The data of the array of sensor readings is used to prepare acoverage map, which is output in step 57 so it can, for example, beviewed by a technician as the puddle changes.

The results of the coverage map scanning 55 may be also used in asoftware-implemented control loop to automatically control the fluidsupply mechanism that supplies the fluid forming the puddle 11 on theslide, so that fluid continues to be supplied until the predetermineddesired coverage area or the entire slide is completely covered. Thisallows efficient application of the fluid to the entire slide by acompletely automatic process.

Fluid Identification

In addition to purely sensing the presence of the puddle on the surfaceof the slide, it has been discovered that different types of fluids usedin testing or staining a sample have different dielectric coefficientsor properties that make it possible for the sensor array 17 to identifythe specific fluid or fluids that are present in the puddle based on thedielectric or impedance properties that the fluid exhibits responsive toalternating currents with varying frequencies. During the loading offluid onto the slide 1 or afterwards, the sensor array 17 may thereforebe used to determine the specific fluid components in the fluid on theslide to classify what exactly is being applied to the slide. This isaccomplished in the preferred embodiment by polling the plates 19 andmeasuring the impedance or other related electrical property of theindividual capacitor plates 19 responsive to AC current with a voltageof 1 to 10 volts at a set of, e.g., five, discrete fingerprintfrequencies which are developed for the given fluids to be applied.

The measurement at different frequencies allows the system todiscriminate between different types of fluids that are applied whetheralone or mixed together with other fluids. This measurement is made atstep 57. A logistic regression is applied to classify the identificationof the fluid in step 59 and an output report indicating theidentification of the reagent is made at step 61.

Generally, aqueous fluids provide for a greater passage of voltagethrough the capacitor plate 19 due to the polarity of the solution andits ability to conduct electricity to some degree. In contrast, oils andother organic solvents or reagents tend to have higher impedances, dueto a greater resistance to conduction of the electrical current throughthem. In addition, different solutions have different levels ofdielectric dependent on the different frequencies being applied. Thefrequencies of the AC current applied generally start at about 12 KHz,and increase from that level. For example, a set of frequencies of 16.3kHz, 22.3 kHz, 1.02 MHz, 2.59 MHz and 3.95 MHz may be advantageouslyemployed in the system.

Referring to FIG. 10, it may be seen that over a range of frequencyinputs from one to ten thousand kilohertz or kHz, the effect ofdifferent fluid compositions on the input to output voltage ratio is avariable and to a degree distinctive for each of the exemplary fluids,i.e., bluing solution, hematoxylin solution, eosin, a differentiatingsolution, a wash solution and air. The ratio of voltage in to voltageout starts at approximately 0.5 and increases up until approximately 10kHz, whereupon a plateau is reached at different levels for thedifferent compositions of fluid, and then, subsequently, the impedanceof the given fluids results in the differing levels of output voltageover a range of frequencies.

Specific fluids may be identified by checking their varying impedance ordielectric qualities at five different distinct frequencies starting atapproximately 12 kHz. A neural net using these frequency values andinputs in voltage and voltage out ratios is used to train a system todevelop a fairly high reliability in terms of identifying the specificfluid composition that is applied to the slide.

One use for this determination is to determine if the wrong fluid isused, and to alert a human user of the apparatus responsive to such adetermination.

Volume Estimation

Returning to FIG. 8, once the coverage map has been completed, it ispossible for the software to further calculate a volume estimate in step63. Volume is determined for the amount of fluid that is present on theslide by projecting the volume based on the data indicative of the areaof the slide and the coverage of the slide, i.e., complete coverage or alesser amount, combined with the defined fluid composition data. Fromthis data, the thickness of the puddle and its general dimensions can bedetermined, from which an estimate of the volume of fluid present can becalculated.

This volume estimate data value is output at 65 to indicate the volumeof fluid that has been applied to slide 1.

Once these determinations and/or outputs have been made, a determinationis made whether to proceed with the analysis or to end the experiment atstep 67, returning the control to the original initial setup, step 51,where a new slide with a new sample is loaded into the apparatus and abaseline threshold impedance for the slide with only the sample isdetected.

Referring now to FIG. 9, in an alternate embodiment, a slide 71 isitself provided with an embedded sensor array. The slide 71 of thisembodiment has a top plate or film 73 that overlies a sensor array ofplates 75, which are electrically connected as for the plates 19 of theprevious embodiment. The slide 71 has a row of contacts 77 on one longedge and one short edge of the slide, which provide for contact of theinternal lines, similar to lines 22 and 24 of the previous embodiment,connecting the plates 75 with the electronic interrogation circuitry ofthe sensing hardware, so that when the slide is inserted into the trayin a base structure 3 in the apparatus 5, the electrical contacts aremade, and operation of the coverage of the slide and its sample with thefluid puddle overlying all of the sensors is possible.

The terms herein should be read as terms of description rather than oflimitation, as those with this disclosure before them will be able tomake changes and modifications without departing from the spirit andscope of the disclosure.

1. A system for detecting fluid coverage of a substrate, comprising: a.a substrate holder for holding the substrate; b. an array of capacitiveplates in proximity to the substrate holder; c. a fluid supplyconfigured to deliver a fluid to the substrate held on the substrateholder; and, d. sensing electronics in electrical connection to thearray of capacitive plates, wherein the sensing electronics periodicallyconnect two poles of a current to at least one pair of plates anddetects an output signal indicative of an electric property between theat least one pair of plates.
 2. The system of claim 1, wherein the arrayof capacitive plates is integrated into the substrate holder.
 3. Thesystem of claim 1, wherein the array of capacitive plates is integratedinto the substrate.
 4. The system of claim 1, wherein the array ofcapacitive plates is configured to be held in a position on a side ofthe substrate opposite the substrate holder.
 5. The system of claim 1,wherein the sensing electronics are configured to determine a baselineoutput signal indicative of a substrate having no fluid coveragethereon.
 6. The system of claim 1, wherein the sensing electronics areconfigured to develop a coverage map of the fluid as it is dispensed tothe substrate by detecting a difference in the electric field due to thepresence of the fluid in the vicinity of a plurality of pairs ofcapacitive plates in comparison to the baseline output signal indicativeof the substrate having no fluid coverage thereon.
 7. The system ofclaim 6, wherein the coverage map is provided to a controller thatcontrols the fluid supply to establish a feedback loop that directs thefluid supply to apply additional fluid to the substrate until apre-determined coverage of the substrate is reached.
 8. The system ofclaim 6, wherein the coverage map is used to determine that a fluid hasbeen removed from the substrate by comparing the baseline output signalto a second output signal after the fluid has been removed to determineif fluid remains on the substrate.
 9. The system of claim 1, wherein thecurrent of the sensing electronics is an AC current.
 10. The system ofclaim 9, wherein the AC current of the sensing electronics is scannedover multiple frequencies for detection of the identity of the fluid.11. The system of claim 1, wherein the electric property detectedbetween the at least one pair of capacitive plates is one or more of avoltage, a current, a capacitance, and an impedance.
 12. A method,comprising: a. providing a sample support having a sample thereon to betreated with a reagent fluid; b. applying the reagent fluid to thesample support; and, c. sensing the presence of the reagent fluid on thesample support, said sensing comprising detecting a respective impedancefor at least one pair of a plurality of sensor elements positioned inproximity to the sample support such that presence of the reagent fluidin a respective subarea of the sample support alters the impedance of acapacitor formed between the at least one pair of the plurality of thesensor elements.
 13. The method of claim 12, wherein the sample supportis a slide having a biological sample adhered thereto.
 14. The method ofclaim 12, wherein a degree of fluid coverage is determined from saidsensing.
 15. The method of claim 14, wherein applying the reagent fluidcomprises applying the reagent fluid from a fluid supply mechanism thatis in feedback control with the sensing step such that the reagent fluidis dispensed, mixed or otherwise dispersed to achieve a predetermineddegree of fluid coverage on the sample support.
 16. A method foridentifying a liquid dispensed to a surface, comprising: positioning asensor array in the vicinity of a substrate, the substrate comprisingthe surface to which the liquid is dispensed, the sensor comprising anarray of capacitor plates; measuring an electrical property of at leastone capacitor plate responsive to alternating currents at one or more ofa plurality of different frequencies; identifying the liquid dispensedon the surface of the substrate that is present between the at least onepair of capacitor plates based on the measured electrical property atthe one or more of the plurality of different frequencies.
 17. Themethod of claim 16, wherein identifying comprises identifying the liquidbased on a logistic regression.
 18. The method of claim 16, whereinidentifying comprises identifying the liquid based on a trained neuralnetwork.
 19. The method of claim 16, wherein measuring comprisesmeasuring the impedance of the at least one capacitor plate responsiveto AC current with a voltage of 1 to 10 volts at each of the pluralityof different frequencies.
 20. The method of claim 16, wherein theplurality of different frequencies comprises a plurality of differentfrequencies over a range from one to ten thousand kilohertz.